Currently there are no successful vaccines for the prevention of feline immunodeficiency disease and feline infectious peritonitis disease in cats. Current feline leukemia virus vaccines are available, but their level of efficacy remains questionable and in some cases may cause the disease. Therefore, there is a need in the art for agents and compositions that provide protection from these and other diseases where there is not yet an existing vaccine or that improve the efficacy of existing and commonly used vaccines. In addition, vaccination of kittens is difficult due to inability to overcome maternal antibodies in kittens. Safe and effective agents to help overcome these barriers are also needed.
The stimulation of T-cell activation and proliferation in response to disease in the host is believed to be dependent on two interactions: the recognition of the T-cell receptor (TCR) with immunogenic peptides in the context of the MHC class I molecules and the secondary interaction of accessory ligands, such as CD80 and CD86, with their coreceptors, CD-28 and/or CTLA-4 on the T-cell. The successful interaction of these two pathways leads to activation and proliferation of both CD4+ and CD8+ T-cells and the increased production of Th1 and Th2 type immune regulating cytokines. In the absence of adequate co-stimulation of T-cells, an anergic state may develop, whereby T cells fail to proliferate and secrete cytokines. Over the years, two molecules have emerged as key regulators of T cell responses, CD28 and its ligands, CD80 and CD86. CD28 is the primary T-cell co-stimulatory receptor and upon interaction with CD80 and CD86, it enhances T-cell proliferation and cytokine synthesis, preventing T-cell death. CTLA-4 (also called CD152), a CD-28 homologue, also plays an important role in co-stimulation. Although, not completely understood, it appears to inhibit T-cell costimulatory responses. The interaction and interplay among CD28, CTLA-4 and their ligands CD80 and CD86 in co-stimulatory processes is key to the overall induction and suppression of immune responses to disease in the host. By manipulating the expression of these 4 costimulatory molecules, it may be possible to regulate T-cell responses, through augmentation, suppression or redirection, to raise a desired immune response towards a particular pathogen or disease condition. In particular, they may be useful for vaccination against infectious diseases, treatment of infectious diseases, and treatment of neoplastic, degenerative, autoimmune, and immunodeficiency conditions.
T-lymphocytes of the mammalian immune system display both control and effector functions. T cell progenitors arise in the bone marrow from stem cells and migrate to the thymus. In the thymus, maturation and selection take place to produce a naive population of immune cells that is able to recognize antigen in the context of major histocompatibility complex (MHC) presentation but is not autoreactive. Following thymic maturation, each T cell possesses a clonally distributed T cell receptor (TCR) which determines its antigen specificity. Further, CD4+ and CD8+ T-cells, the two major subsets found in most adult mammals, possess TCR composed of α and β subunits (Allison and Lanier, 1987).
Protein and gene organization of the TCR protein is similar to that observed with immunoglobulin (Ig) molecules, and it shares many properties similar to membrane bound Ig on B cells (Allison and Lanier, 1987). Like the Ig molecule, the TCR must potentially recognize a vast number of potential antigen sequences. For this reason, TCR gene organization and rearrangement is similar in complexity with that observed in B cells (Davis and Bjorkman, 1988). As with antibody molecules produced by B cells, generation of idiotypic diversity in T-cells involves multiple copies of variable (V) genes in the germ line, random rearrangements of a and β subunits, and variability generated by junctional and insertional events (Davis and Bjorkman, 1988). Unlike B cells however, T-cells do not appear to generate diversity through somatic mutation, though the potential repertoire of the TCR appears to be as great as that of the Ig molecule (Lechler et al., 1990).
The TCR, though responsible for antigen recognition, does not have signal delivery capabilities (Allison and Lanier, 1987). Conformational changes in the TCR, following binding to antigen presented in the context of MHC on the antigen presenting cell (APC), result in signal delivery through a noncovalently associated complex of surface molecules including CD3 and the ζ chains (Clevers et al., 1988). TCR binding results in the phosphorylation of the CD3 complex, which indirectly leads to a Ca+ influx into the cell, initiating IL-2 and IL-2R production (Weiss and Littman, 1994). This cascade is considered an initial event in T cell activation.
The TCR recognizes antigen only when it is presented in association with the MHC. There are two subsets of MHC proteins associated with antigen presentation to the T cell. MHC class I is found on almost all nucleated cells within the body and functions to surface express endogenously produced peptides (Matasumura et al., 1992). Peptide expressed in the context of MHC class I is recognized by T-cells expressing CD8 in association with the TCR (Littman, 1987). CD8+ T-cells function in immuno-surveillance for removal of virally infected cells and malignancies. Recognition of non-self molecules by the CD8+ T cell (peptides or altered self peptides that might indicate a malignancy) result in the cytotoxic T-lymphocyte (CTL) mediated destruction of the cell (Berke, 1994).
MHC class II molecules, the second major histocompatibility subset, are normally found only on professional antigen presenting cells including B cells, macrophages/monocytes and dendritic cells, though induction on some other cell populations in response to specific stimuli is possible (Germain, 1993). The MHC class II molecule is responsible for the presentation of exogenous antigen to the CD4+ T cell. Antigen that is phagocytosed, endocytosed or surface Ig bound and absorbed by antigen presenting cells is endogenously processed and bound to MHC class II (Unanue, 1987). The molecule is then surface expressed and available for recognition by CD4 expressing αβ T-cells (Littman, 1987). Antigen recognition by CD4+ T-cells results in the production of cytokines and growth factors necessary for the initiation and promulgation of many facets of an active immune response (Mosmann and Coffman, 1987).
CD4 and CD8 differentiate the αβ T cell subsets and define the functional properties of each group. The presentation of CD4 or CD8 on a T cell is mutually exclusive (Littman, 1987). Thus, following thymic selection and maturation, αβ T-cells present only CD4 or CD8. The molecules act to stabilize the interaction between the TCR and antigen bound MHC and determine whether the T cell recognizes antigen presented in the context of MHC class I or class II (Littman, 1987). The binding domain of the CD4 or CD8 molecule recognizes respective non-polymorphic regions of the class I or class II molecule (Clayberger et al., 1994). Binding of CD4 or CD8 to these specific regions acts to stabilize the TCR/antigen bound MHC interaction, for the initiation of T cell activation (Littman, 1987). Thus, CD4+ T-cells only functionally interact with APC expressing antigen in the context of class II and initiating a T helper response, while CD8+ T-cells only recognize antigen presented in the context of class I, and upon binding initiate a cytotoxic response (Germain, 1993). The two distinct phenotypes of helper T-cells and CTL can be differentiated by the surface expression of either CD4 or CD8.
The majority of T-lymphocytes bearing CD4 are generally considered to be helper cells though there is a proposed CD4+ CTL subset (Yasukawa, et al., 1989). CD4+ helper T-cells are major regulators of the immune response through the production of a battery of stimulatory and suppressive cytokines (Mosmann and Coffman, 1987). The factors produced by these cells are important mediators in the initiation of both a humoral or antibody mediated response and a cellular or delayed type hypersensitivity (DTH) response (Mosmann and Coffman, 1987). For CD4+ T-cells to become activated and produce soluble growth factors, a complex cascade of events must occur. Antigen is detected and endocytosed by a professional APC, normally a macrophage (Unanue, 1984). The APC denatures the protein and breaks it down into smaller fragments, peptide fragments of between 15-18 amino acid residues are then bound with the MHC in the endoplasmic reticulum and subsequently transferred to the surface for expression (Rotzschke et al., 1994). Surface expressed antigen is thus visible to T-lymphocytes and can be recognized by the T cell subsets with the proper TCR idiotype and expressing CD4 (Germain, 1993). When the T cell recognizes the proper antigen, and the proper accessory signals are delivered, differentiation of the naive lymphocyte occurs and clonal expansion can proceed. As yet undetermined stimuli result in the preferential development of a type 1 (cellular) response versus a type 2 (humoral) response (Mosmann and Coffman, 1989).
T cell help is required for much of the activity of both a humoral and a cellular response. A T cell dependent B cell response, which is required for antibody to be made to most antigens, requires T cell help for proper B cell maturation to take place (Chesnut et al., 1986). Once the surface expressed Ig on the B cell has bound antigen, internalization, processing and MHC class II surface expression of these antigens occurs (Germain, 1993). Direct cell to cell contact between the CD4+ T cell with the proper TCR idiotype and the B cell, promotes the activation and proliferation of the T cell (Chesnut et al., 1986). The activated helper T cell may be capable of promoting a type II response by secreting factors necessary for B cell growth and differentiation (Mosmann and Coffman, 1989). These factors include, IL-4, IL-5 and IL-13 which can induce B cell activation and proliferation, and are important in isotypic switches for the Ig molecule, while IL-10 acts to prevent the initiation of a type I response which would in turn down-regulate humoral activity (Mosmann and Coffman, 1989).
Cellular responses (type I) do not mature in the same fashion as humoral responses (type II) (Sher et al., 1992). Upon T cell activation and maturation to a type 1 response, factors are produced by the T cell that favor cellular immunity. IL-2 is a T cell growth factor that also promotes CTL responses, while IFNγ acts to activate macrophages, CTL and neutrophils (Wang et al., 1993).
T helper cells are thus able to mediate two largely mutually exclusive responses. The cytokine secretion pattern that leads to the initiation of a humoral response contains factors that are suppressive of a cellular response and vice versa (Mosmann and Coffman, 1989). It is unclear what determines whether a T cell will produce a type 1 pattern (IL-2, IFNγ and lymphotoxin) or a type 2 pattern (IL-4, 5, 6, 10, and 13) although it is proposed that the type of APC that presents the antigen or soluble factors produced by the APC may influence the type of cytokine pattern that develops (Mosmann and Coffman, 1989). In addition to the type 1 and type 2 helper cells, T helper type 0 subsets exist in which secretion patterns are intermediate between type 1 and type 2 (Gajewski et al., 1989). While T helper subsets have mainly been demonstrated in in vitro experimentation, and may be artifacts of culture, they are important models for the role of the T helper cell in modulating the development of specific responses in an in vivo environment.
In addition to the CD4+ T helper lymphocyte subset, a second αβ T cell population consists of CD8 bearing cytotoxic lymphocytes. The CD8+ CTL appear to be a major component of the immune surveillance system whose primary function is to destroy virally and intracellular bacterially infected cells as well as malignancies (Berke, 1994). These cells are also able to produce cytokines, but generally only those associated with inducing cellular responses (IL-2, IFNγ and TNF) (Fong and Mosmann, 1990). The TCR of these cells, in association with CD8, recognize antigen presented in the context of MHC class I (Littman, 1987). In general, all nucleated cells have surface expression of class I presenting endogenously synthesized peptides (Matasumara, 1992). Specific, immuno-privileged sites, including the brain and the testes, have low level expression of the protein, though it is inducible in these areas with interferon exposure (Moffett and Paden, 1994).
Proteins produced in the endoplasmic reticulum through the normal metabolism of the cell are denatured, partially degraded and bound to MHC class I for surface expression (Engelhard, 1994). The polypeptides are proteolytically linearized and bound in 9-12 amino acid epitopes to class I which is then expressed on the surface of the cell (Engelhard, 1994). Theoretically all endogenously produced peptides are surface expressed in this fashion, and thymic selection ideally results in the elimination of all autoreactive T-cells, immune-surveillance can detect the presence of virally infected or transformed cells (Berke, 1993). The recognition of foreign peptides expressed by class I is facilitated by the antigen specific T cell receptors on CD8+ CTL (Lechler et al., 1990). Contact between the effector cell and the target is required for activation to proceed (Berke, 1994). When an antigen that is seen as foreign is detected by the TCR, the interaction between the molecules is stabilized by CD8 binding to the class I on the infected cell (Littman, 1987). Once recognition occurs and the T cell becomes activated, a conjugate forms between the target cell and the effector T cell, and the effector cell is dispatched (Taylor and Cohen, 1992). Thus, in this fashion, if self proteins are altered or if the cellular machinery is taken over by a pathogen, peptides will be available for recognition by immune surveillance and this arm of the immune system can eliminate the diseased cell (Berke, 1994).
Cellular cytotoxicity appears to result from one of two major pathways. Either the cell is induced to undergo apoptotic death or it is lysed through the release of cytotoxic granules by the CTL (Berke, 1993). Apoptosis is induced in target cells through the release of factors by the CTL which induce gene expression that result in cell death (Russel, 1983). An advantage of this mechanism is that cell lysis does not occur and the potential for the release of the potentially infectious contents of the cell is reduced (Nagata and Golstein, 1995). Cell lysis however may be the more common mechanism through which target elimination takes place. Perforin, which acts to perforate target cell membranes, is a major constituent of CTL cytotoxic granules (Liu et al., 1995). Although there are other cell types involved in this form of immuno-surveillance, CTL appear to be a major component of anti-viral and anti-tumor immunity and, against specific pathogens, are considered indispensable for protection (Kupfer and Singer, 1989).
Initially cell surface proteins were used to differentiate specific cell populations. More recently functional aspects of many of these molecules have been derived, and while they are still important in delineating cell populations, their critical role in the function of many cells is becoming more evident.
A variety of accessory and adhesion molecules that play a role in the development of a productive immune response are expressed on T-cells and antigen presenting cells (van Seventer et al., 1991). Adhesion molecules are expressed at some level on most cells of the immune system. They are important in retaining cells within an area and in the initiation and maintenance of cell to cell contact (Mescher, 1992).
CD-2/LFA 3 (CD58) and LFA-1/ICAM-1 are two adhesion molecule complexes involved in the stabilization of T cell/APC interactions and the enhancement of activity (Springer et al., 1987). CD2 is one of the first markers expressed on pre-T-cells and persists throughout the life of the cell, while LFA-1 is expressed later on T-cells and is up-regulated in memory cells or by induction (Springer et al., 1987).
Accessory molecule complexes also demonstrate adhesive properties, but their main function is probably the delivery of an intracellular signal upon ligand binding (Anderson et al., 1988). Upon establishment of an interaction between the receptor and its ligand, a conformational change in the molecules structure takes place that results in the delivery of a signal to the cytoplasm of one or both cells (Hutchcroft and Bierer, 1994). Signals delivered by these molecules play a variety of roles in promoting T cell development, but in the absence of signals mediated by these molecules, T-cells may become anergic (Leung and Linsley, 1994).
The CD28/CD80 interaction is a major component of a productive T cell mediated immune response (Linsley et al., 1993a). The interaction of the CD28 accessory molecule with its ligand CD80 is required for full activation and proliferation of naive T-cells (Linsley et al., 1991a). The interaction also appears to play a critical role in activated and memory CD4+ T cell proliferation and prevention of apoptotic cell death (Linsley et al., 1991a). The discovery of the interaction and elucidation of its mechanisms has provided a critical link in the understanding T cell mediated immunity.